THYMOSIN ALPHA 1, A NEW BIOLOGIC RESPONSE MODIFIER FOR THE TREATMENT OF VIRAL HEPATITIS AND HEPATOCELLULAR CARCINOMA
Eduardo B. Martins, M.D.* Ph.D., Cynthia Tuthill, Ph.D.*
La timosina alfa-1 es un nuevo inmuno modificador de la respuesta biológica que ha sido investigado en humanos para el tratamiento de las hepatitis crónicas por virus B y C y para algunos tipos de cáncer, especialmente el carcinoma hepatocelular. Todos estos aspectos son revisados en este artículo.
Palabras clave: Timoshina alfa-1, tratamiento hepatitis crónica por virus B, hepatitis crónica por virus C, carcinoma hepato celular.
Thymosin alpha-1 is a new biologic response modifier that has been investigated in humans for treatment of chronic hepatitis B, chronic hepatitis C, and some types of cancer, particularly hepatocellular carcinoma. All these particular aspects are here reviewed.
Key words: Thymosin alpha-1, treatment chronic hepatitis B, treatment chronic hepatitis C, hepatocellular carcinoma.
Thymosin alpha-1 (Ta1), originally
isolated from the thymus gland and currently produced
Ta1 has been shown to have a number of immunomodulatory activities centered primarily on augmentation of T-cell function (Table 1).
Many of the in vitro and in vivo effects of Ta1 have been interpreted as influences on either differentiation of pluripotent stem cells to thymocytes and/or activation of thymocytes into activated T-cells. The pattern of enhanced cytokine production, i.e., IFNg and IL-2, suggest that Ta1 may influence progression to a Th1 type of immune response .
Although Ta1 seems to act primarily through its immunological actions, recent experiments have shown that Ta1 also has antiviral effects. In duck hepatocytes infected with duck hepatitis B virus, Ta1 treatment significantly reduced viral replication, especially at the level of expression of intrahepatic viral proteins . Ta1 was also shown to be effective in the woodchuck hepatitis model for CHB. In a study with 36 animals given Ta1, serum WHV DNA levels fell by 99-99.9% in the treated groups compared to no change in the control animals . These results are similar to those obtained with IFNa . Finally, Ta1 has been shown to increase the expression of MHC class 1 in cultured cells thus being capable of increasing antigenicity of virally-infected cells (manuscript in press).
It should also be noted that many effects of Ta1 appear to be synergistic with those of other cytokines, and the peptide may work best in combination with other immunomodulators. For example, Ta1 enhanced the effects of IFNa on the NK-cell activity of lymphocytes either from mice , normal human donors  or from HIV-infected patients . Ta1 also augments the effects of IL-2 in lymphocytes from both normal and immune suppressed mice .
Given its immunoregulatory properties, Ta1 has been investigated in humans for treatment of chronic hepatitis B (CHB), chronic hepatitis C (CHC), and some types of cancer, particularly hepatocellular carcinoma (HCC). Furthermore, in treatment of over 3000 patients in clinical trials worldwide with a range of diseases including hepatitis B and hepatitis C, Ta1 has been well tolerated and was not associated with virtually any significant side effects.
CHB is a widespread disease associated with significant morbidity and mortality. At least 300 million people worldwide are chronically infected with the hepatitis B virus (HBV), with carrier rates as high as 20% in some populations. CHB is associated with increased risk for cirrhosis, liver failure and hepatocellular carcinoma. Impaired effectiveness of the host cellular immune mechanisms in clearing HBV-infected hepatocytes has been proposed to explain development of chronic HBV infection.
In clinical studies for CHB, Ta1 has been primarily investigated as monotherapy, but promising results have also been obtained when the peptide is used in combination with nucleoside analogues.
In a study from Taiwan, 98 patients with vertically-transmitted, CHB were randomly allocated to 3 groups: 1) group A received a 26-week course of thymosin alpha 1 with a 1.6-mg subcutaneous (sc) injection two times a week; 2) group B received the same regimen as group A, but thymosin alpha 1 therapy extended for 52 weeks; and 3) group C served as a control group and was followed up for 18 months without specific treatment. The three groups were comparable in clinicohistological features at entry. The complete virological response rate (clearance of serum HBV DNA and HBeAg) was higher in group A (40.6%) and group B (26.5%) than in group C (9.4%) (group A vs. group C: P=0.004; group B vs. group C: P=0.068) when assessed 18 months after entry, although complete response rates among these three groups were similar when first assessed at the end of therapy. There was a trend for complete virological response to increase or accumulate gradually only after the end of thymosin alpha 1 therapy, whether that therapy was given over 26 months or 52 weeks. The 34 patients in Group B were evaluated again at the end of 24 monthsi.e., 12 months after their Ta1 treatment had ended. In that 6-month period, the number of responders rose from 9 (26.5%) to 13 (38.2%). These results are summarized in Figure 1.
These results reflect the typical pattern of Ta1 efficacy that has been demonstrated in earlier studies: response rates increase continuously for at least 12 months after treatment has ended. Blinded histological assessment showed a significant improvement in treated patients, particularly in lobular necroinflammation and scores excluding fibrosis. No significant side effects were observed. These results suggest that 26-week and 52 week courses of thymosin alpha 1 therapy are effective and safe in patients with chronic hepatitis B .
In a multicenter, randomized, controlled trial Andreone et al. investigated the efficacy of Ta1 versus IFNa2b in 33 patients with pre-core mutant CHB (HBV DNA and anti-HBe antibody positive). Sixteen patients received IFNa2b (5 MIU, three times weekly) and 17 patients received Ta1 (0.9 mg/m2, two times weekly) for 6 months and were followed for an additional 6 months. The results were further compared to a group of 15 matched untreated historical controls followed for 12 months. Complete response was defined by ALT normalization and HBV DNA loss after the 6-month follow-up period.
Complete response occurred in 41% (7/17) in the Ta1 group and 25% (4/16) in the IFNa2b group (p=ns). Compared with results observed in 15 untreated patients, the rate of complete response was significantly higher in the Ta1 group (41% versus 7%, p<0.05, Fishers exact test). The IFNa2b response was not significant when compared to control, due to a high relapse in the IFNa2b patients during the follow-up period after treatment was completed. Unlike IFNa2b, therapy with Ta1 was not associated with side effects. 58% of the Ta1 -treated patients showed histological improvement after the 6-month follow-up, 25% were unchanged, and 17% worsened. The IFNa2b -treated patients showed 36% improvement in liver histology, 36% unchanged, and 27% worsened.
The data from this trial illustrate an important apparent difference between Ta1 and interferon treatment. With interferon, responses are typically seen during the first four months of therapy, with relapses in the follow-up period. In this trial, the response rate in the IFNa2b group declined from 44% at the end of the 6-month treatment to 25% at the end of the 6-month follow-up. With Ta1, responses continue to occur after treatment is stopped (in this trial, 29% at the end of the 6-month treatment and 41% at the end of the 6-month follow-up). This delayed response to Ta1 treatment has also been seen in the Taiwan trial .
Nucleoside Analogues and Ta1 Combination Therapy in Hepatitis B
The response rate to interferon in Asian patients is low: only 15-20% will clear HBeAg and HBV DNA. One of the factors that decreases efficacy of an immunomodulatory agent is a high pre-treatment HBV DNA level. Recently, second generation nucleoside analogues such as lamivudine and famciclovir have been shown to be effective in suppressing HBV replication. It would therefore be logical to use a combination of immunomodulatory agents and second generation nucleoside analogues in the treatment of chronic HBV.
Leung and So used Ta1 and a combination of two nucleoside analogues, lamivudine and famciclovir, in 11 Chinese patients with CHB and high levels of HBV DNA . Patients received the three drugs for 6 months, followed by lamivudine and famciclovir for a further 6 months. The endpoint was a complete virological response defined as disappearance of HBV DNA and HBeAg. After 12 months of follow-up, 64% of patients had a complete virological response. Furthermore, all patients normalised their (alanine aminotransferase) ALT and all but one patient became HBV DNA negative.
This data suggests that a combination of Ta1 and nucleoside analogues may be a safe and even more effective therapy for CHB, and larger studies are currently ongoing in the USA.
CHC is recognized as a global health problem, with an estimated worldwide prevalence of over 200 million and no foreseeable vaccine. Up to 85% of patients infected with HCV go on to chronic infection, with a high incidence of liver disease, including cirrhosis and hepatocellular carcinoma. Clearance of HCV infection requires immune involvement, although the exact mechanism for clearance may be different from that seen in HBV infection.
Three studies have investigated the therapeutic effect of Ta1 in combination with IFN for treatment of chronic hepatitis C. These are a phase 3 study in the United States  and two phase 2 studies in Italy [31, 32]. These studies have been analyzed independently, and using pooled and meta-analysis techniques.
Sherman et al.  conducted a randomized, placebo-controlled, double-blind, multicenter trial comparing Ta1 (1.6 mg, sc, two times weekly) plus IFNa2b (3 MIU, sc, three times weekly) combination treatment to IFNa2b alone or placebo. 110 patients were enrolled and randomized to one of the three treatment groups; IFNa2b alone, Ta1 plus IFNa2b combination, or placebo. The treatment period was 6 months. After 6 months, the responders were followed for an additional 6 months. Patients who did not respond to IFNa2b monotherapy or combination therapy were offered a 6-month combined regimen of Ta1 plus IFNa2b. The primary clinical endpoint was complete biochemical response, defined as a normal ALT level on the last two study visits at the end of the 6-month treatment period.
For the 107 evaluable patients, there was a greater complete biochemical response in the Ta1 plus IFNa2b combination group (42%, 14/33) compared to IFNa2b (19%, 7/37), and placebo (5%, 2/37) at the end of treatment. Among the three treatment groups, the IFN plus Ta1 combination group was statistically significantly different from placebo and IFN alone (p<0.001 and p=0.04, respectively). Partial responders were considered in the efficacy analysis as non-responders.
All patients treated with IFNa2b alone who responded to treatment, responded by 12 weeks. In contrast, patients receiving Ta1 plus IFNa2b combination therapy continued to demonstrate responses throughout the study, demonstrating that the Ta1 plus IFNa2b combination enhances later treatment response than typically seen with IFN alone.
An intent-to-treat analysis of viral clearance (end-of-treatment viral load compared to viral load at baseline) demonstrated a significant difference between the Ta1 plus IFNa2b and placebo groups (p<0.001), and a statistical trend in the difference between Ta1 plus IFNa2b and IFNa2b (p=0.1) in HCV RNA levels. Patients with Knodell histological activity index score improvement of more than 2 points in the Ta1 plus IFNa2b, IFN, and placebo groups were, respectively, 47% (16/34), 36% (12/33) and 14% (5/36) (p=0.01 among the three treatment groups; p=0.004 between Ta1 plus IFNa2b and placebo). Improved histological activity index was associated with ALT response. After 6 months of therapy, 11 patients who did not respond to IFNa2b were placed on a 6-month combined regimen of Ta1 plus IFNa2b. Ten of the 11 patients completed 6 months of the combined regimen. After 6 months, 40% (4/10) showed normalization of ALT. Eight patients (80%) demonstrated at least a 50% decrease in viral titer with three (30%) patients classified as complete virological responders. The authors concluded that Ta1 plus IFNa2b combination therapy was superior to single-agent IFNa2b in treatment of patients with CHC, and was generally well tolerated.
Two studies in Italy have also investigated Ta1 plus IFN combination therapy for the treatment of chronic hepatitis C. Moscarella et al.  conducted a randomized study to compare the efficacy of Ta1 plus IFNa2b combination treatment to IFNa2b alone. Patients had histologically proven HCV-positive chronic active hepatitis and persistent mean ALT > 2 times upper limit of normal, and had not been previously treated with any drug for their disease. Seventeen patients were randomized to receive IFNa2b (3 MIU three times weekly) and Ta1 (2 mg two times weekly) for 6 months, and 17 patients received IFNa2b alone (3 MIU three times weekly). All patients were followed for 12 months after treatment ended (total 18 months). After the 6 months of therapy, complete response (defined as normal ALT at the end of therapy) was observed in 71% (12/17) of the patients treated with Ta1 plus IFNa2b combination therapy and 35% (6/17) of the patients treated with IFNa2b alone (p=0.04). Sustained response at 18 months was 29% (5/17) in the Ta1 plus IFNa2b combination group and 18% (3/17) treated with IFNa2b alone.
Rasi et al.  conducted an open-label phase 2 study of CHC patients using a combination of Ta1 and lymphoblastoid IFNa (L-IFNa). Treatment consisted of one week of inductive therapy (1 mg Ta1, sc, on days 1-4 and 3 MIU L-IFNa, im, on day 4) followed by maintenance treatment from weeks 2 to 52 (1 mg Ta1, sc, two times weekly and 3 MIU L-IFNa, im, three times weekly). Patients were followed for an additional 6 months after completion of the 12-month treatment period (18 months total). Response was defined as negative serum HCV RNA by PCR at 12 months. Sustained response was defined as negative serum HCV RNA by PCR after 6 months follow-up. Fifteen patients were entered into the study, including four who had failed previous IFNa therapy. Thirteen of the 15 patients were of genotype 1b, the genotype least responsive to interferon therapy. Six patients had active cirrhosis. After completion of 12 months treatment, 73% (11/15) including two of the four IFNa2b failure patients responded with loss of serum HCV RNA; eight of these patients also responded with normal ALT. Sixty-nine percent (9/13) of the patients with HCV type 1b treated with the IFNa2b and Ta1 combination responded to therapy and five (39%) were still negative for serum HCV RNA 6 months after treatment ended. Genotype 1 response to retreatment with interferon and ribavirin, by comparison, has only a 28% response rate . Moreover, at 18 months, 50% (3/6) of the patients with active cirrhosis, a more difficult to treat population, responded to therapy with a loss of HCV RNA, and two patients (33%) were still negative 6 months after treatment ended. The overall sustained response (loss of serum HCV RNA after 18 months) was 40% (6/15). Five of the six HCV RNA negative patients at 18 months also had normal ALT levels. Patients with a sustained response to treatment showed significant improvement in the histological activity index after treatment (p<0.05). No major toxicity was observed and no patient reduced IFN dosage or suspended treatment. In this study, combination treatment for 12 months resulted in a greater sustained response rate over that seen with 6 months treatment in other trials [30, 32].
Pooled and Meta-Analysis Ta1/IFNa Combination Therapy for Chronic Hepatitis C
The 3 studies discussed above have been analyzed independently, and using pooled and meta-analysis techniques .
A total of 136 patients (67 Ta1/IFN combination therapy, 54 IFN monotherapy and 15 monotherapy historical controls) were included in the meta-analysis. A total of 121 patients (67 Ta1/IFN combination therapy and 54 IFN monotherapy) were included in the pooled analysis. In these studies the majority of patients were infected with the difficult-to-treat HCV genotype 1, and a substantial number were cirrhotics.
Pooled intent-to-treat analysis revealed an end-of-treatment (ETR) biochemical response (ALT) of 45% in the Ta1/IFN combination treatment group compared to 22% in the IFN monotherapy group (p=0.0096). Sustained biochemical response, defined as normal ALT 6 to 12 months after completion of treatment, was observed in 9% of patients treated with IFN alone compared to 22% in the Ta1/IFN combination therapy.
The meta-analysis demonstrated a biochemical response odds ratio and 95% confidence interval (CI) of greater than 1, and an odds ratio of 3.1 for the combined studies. This indicates that the combination of Ta1 and IFN was more than 3 times better at normalizing ALT at the end of treatment than IFN alone. This difference was statistically significant. In addition, meta analysis demonstrated a sustained response odds ratio of greater than 1 and a 95% CI slightly overlapping 1, indicating that Ta1/IFN combination therapy was also superior to IFN monotherapy for sustained biochemical response.
For the virological response data, meta-analysis of both end-of-treatment and sustained response (defined as undetectable HCV RNA by PCR at 6 to 12 months after the end of treatment) showed response odds ratio with a 95% CI of greater than 1 for the combined studies, indicating that Ta1/IFN combination therapy was statistically significantly superior to IFN monotherapy.
One study has been completed which examined the efficacy and safety of Ta1 for treatment of hepatocellular carcinoma . Twelve patients with hepatocellular carcinoma (HCC), 11 with cirrhosis of class A or B according to Childs criteria, with tumors classified as Okuda stage I or II, and one patient with class C cirrhosis and an Okuda stage III tumor, were admitted to the trial after a diagnosis of HCC was established based on ultrasonography and histology. The patients were treated with 0.9 mg/m2 Ta1 (sc, two times weekly) for 6 months, and transcatheter arterial chemoembolization (TACE) using 40-60 mg of doxorubicin. Results were compared to a historical control group matched for gender, age, Okuda staging, Childs score, alpha feto-protein serum levels and viral infection, who were treated with TACE alone.
Patients treated with the combination of Ta1/TACE showed longer survival compared to the historical control group treated with TACE alone, that reached statistical significance 7 months after the end of treatment (82% vs. 41% survival; p<0.05, Kaplan-Meier and Mantel-Haenszel test). Moreover, patients receiving the Ta1/TACE combination demonstrated a significant increase in cytotoxic T cells (CD8) and NK cells (CD16 and CD56) at 3 months and 1 month, respectively, after completion of treatment. It is possible that the improvement in immune parameters was associated with the increased survival seen in this study. A larger, multicenter, randomized controlled trial is currently under way to test this hypothesis.
Address for correspondence: